A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

保护哺乳动物生殖系基因组完整性的新工艺

• 批准号: 10445720
• 负责人: Piroska Edit Szabó
• 金额: $ 51.9万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-20 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445720
• 关键词: Adopted; Affect; Affinity; B-DNA; BTB/POZ Domain; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biological Process; Cell Culture Techniques; Cells; Cellular biology; Chromosomal Rearrangement; Chromosome Deletion; Circular Dichroism; DNA; DNA Double Strand Break; DNA Methylation; DNA Modification Methylases; DNA Sequence Rearrangement; DNA Structure; DNA-Binding Proteins; Dependence; Development; EMSA; Enzymes; Epigenetic Process; Generations; Genetic; Genetic Models; Genome; Germ Cells; Germ Lines; Germ-Line Mutation; Grant; Hand; Human; Immunohistochemistry; In Vitro; Left; Location; Mammalian Cell; Maps; Measures; Methods; Methylation; Methyltransferase; Molecular; Monitor; Mus; Mutant Strains Mice; Nucleosomes; Organism; Population; Pregnancy; Process; Proteins; Purines; Pyrimidine; Refractory; Role; Short Tandem Repeat; Site; Spermatocytes; Tandem Repeat Sequences; Testing; Time; Transgenic Mice; Variant; Z-Form DNA; Zinc Fingers; base; epigenome; epigenomics; fetal; genome integrity; genome-wide; in vivo; male; mutant; mutation assay; next generation; novel; sperm cell

PROJECT SUMMARY Simple purine–pyrimidine (Pu/Py) repeats (PPRs) are known to form the left-handed, fragile Z-DNA structure. Such repeats are also known to be highly mutagenic, inducing large chromosomal deletions and rearrangements in the cells of higher organisms. The mutagenic effects of Z-DNA would be the most detrimental to a species if breaks occurred uncontrollably in the germ line and mutations occurring at unrepaired breaks were passed on to the next generation. We discovered a novel biological process in the mammalian germ line that controls Z- DNA structure at PPRs. Using the Zbtb43 mutant mouse genetic model and our transgenic mouse line that allows us to isolate germ cells, we found that a previously uncharacterized DNA binding protein, ZBTB43 remodels Z-DNA structure and protects from double-strand breaks in fetal male germ cells in vivo. By biochemical assays we found that ZBTB43 binds to PPR-rich DNA sites in the genome in vitro. ZBTB43 binding sites form Z-DNA and cause large genomic rearrangements in mammalian cells. By in vivo epigenome mapping we detected Z-DNA in mutant germ cells at the locations where ZBTB43 binding occurs in wild-type prospermatogonia. We hypothesize, therefore, that ZBTB43 safeguards genome integrity in the germ line by binding and eliminating Z-DNA at PPRs. In addition, we found that by eliminating Z-DNA, ZBTB43 promotes de novo methylation at PPRs during the time of global epigenetic remodeling. We propose to pursue the following Aims, using a combination of genetic, cell biology, biochemistry, and epigenomic approaches. In Aim 1, we will test the working hypothesis that ZBTB43 eliminates Z-DNA structure in vivo by directly binding to PPRs in fetal male germ cells. We will determine 1) the spatial and temporal changes of the Z-DNA structure in fetal male germ cells in the presence or absence of ZBTB43 protein in vivo; 2) the dependence of the Z-DNA remodeling process on the direct binding of ZBTB43 to the Z-DNA structure in vivo; and 3) the molecular requirements of ZBTB43 action on Z-DNA. In Aim 2, we will test the working hypothesis that ZBTB43 facilitates de novo DNA methylation in prospermatogonia indirectly by eliminating Z-DNA, thus revealing the sequences as substrates for de novo methyltransferases. We will map DNA methylation in the presence and absence of ZBTB43 during the epigenome remodeling process in fetal male germ cells. We will test whether de novo DNMTs methylate Z- DNA substrates in vitro. We will test whether ZBTB43 affects nucleosome occupancy at PPRs. In Aim 3, we will test the hypothesis that Z-DNA is mutagenic in the germ line and that ZBTB43 has evolved to manage that burden. We will map double-strand breaks in mutant fetal germ cells, test the anti-mutagenic effect of ZBTB43 in cell culture, and search for genomic rearrangements in sperm of Zbtb43 mutant males. By the end of the grant period, we will have identified and characterized the first example of how a DNA binding protein protects genome integrity and targets de novo DNA methylation by controlling Z-DNA structure in the mammalian germ line.

项目摘要 已知简单嘌呤-嘧啶（Pu/Py）重复序列（PPR）形成左手的、脆弱的Z-DNA结构。 这种重复序列也被认为是高度致突变的，诱导大的染色体缺失和重排 在高等生物的细胞中。Z-DNA的诱变效应对一个物种是最有害的， 在生殖细胞系中不受控制地发生断裂，在未修复的断裂处发生的突变被传递 传给下一代我们在哺乳动物生殖细胞系中发现了一种新的生物学过程，它控制着Z- PPR的DNA结构。使用Zbtb 43突变小鼠遗传模型和我们的转基因小鼠系， 在分离生殖细胞的过程中，我们发现一种以前未被鉴定的DNA结合蛋白ZBTB 43， 重塑Z-DNA结构，并在体内保护胎儿雄性生殖细胞免受双链断裂。通过 在生物化学测定中，我们发现ZBTB 43在体外与基因组中富含PPR的DNA位点结合。ZBTB 43结合 位点形成Z-DNA并在哺乳动物细胞中引起大的基因组重排。通过体内表观基因组作图 我们在野生型ZBTB 43结合发生的位置检测到突变生殖细胞中的Z-DNA 双头龙属。因此，我们假设ZBTB 43通过以下方式保护生殖细胞系的基因组完整性： 在PPR处结合并消除Z-DNA。此外，我们发现，通过消除Z-DNA，ZBTB 43促进了细胞凋亡。 全球表观遗传重塑期间PPR的新生甲基化。我们建议采取以下措施 目的是利用遗传学、细胞生物学、生物化学和表观基因组学方法的组合。在目标1中，我们 测试ZBTB 43通过直接结合胎儿中的PPR来消除体内Z-DNA结构的工作假设。 男性生殖细胞我们将确定1）男性胎儿Z-DNA结构的时空变化 ZBTB 43蛋白存在或不存在下的生殖细胞; 2）Z-DNA重塑的依赖性 ZBTB 43与Z-DNA结构在体内直接结合的过程;和3）ZBTB 43的分子要求。 ZBTB 43对Z-DNA的作用。在目标2中，我们将测试ZBTB 43促进从头DNA的工作假设 通过消除Z-DNA间接地在原核细胞中进行甲基化，从而揭示作为底物的序列 对于从头甲基转移酶。我们将在ZBTB 43存在和不存在的情况下绘制DNA甲基化， 胎儿雄性生殖细胞的表观基因组重塑过程。我们将测试从头DNMT是否甲基化Z- 体外DNA底物。我们将测试ZBTB 43是否影响PPR处的核小体占据。在目标3中，我们 测试Z-DNA在生殖系中具有致突变性以及ZBTB 43已经进化来管理这一假设 负担我们将绘制突变胎儿生殖细胞的双链断裂图谱，检测ZBTB 43的抗突变作用 在细胞培养中，并在Zbtb 43突变雄性精子中寻找基因组重排。在补助金结束时 在此期间，我们将确定并表征DNA结合蛋白如何保护基因组的第一个例子 完整性，并通过控制哺乳动物生殖系中的Z-DNA结构来靶向从头DNA甲基化。